Continuing Researchers: Anita V, Serena Z, Oscar V, De La O, Daniel D, Imran Z, Madeline J, Fin G, Grant T, Gautam W
Please fill out a progress report in this format every two weeks regarding where you are in your research. In "Summary," briefly explain what you have accomplished in the last two weeks.
We have a UTList Serv that we can communicate when people will be in lab. Anita will add you to the list. **vds-continuing-researchers**
Bi-Weekly Update Schedule (i.e. every other week, not twice weekly!)
-- copy these lines below to your section
-- update your progress for each 2 week block
-- make a new goal for next 2 weeks Wed - Jan 22nd Progress: Goal for Next 2 Weeks:
Wed - Feb 5th Progress: Goal for Next 2 Weeks:
Wed - Feb 19th Progress: Goal for Next 2 Weeks:
Wed - Mar 5th Progress: Goal for Next 2 Weeks:
Wed - Mar 19th Progress: Goal for Next 2 Weeks:
Wed - April 2nd Progress: Goal for Next 2 Weeks:
Wed - April 16th Progress: Goal for Next 2 Weeks:
Wed - April 30th Progress: Goal for Next 2 Weeks:
Joey 1/22/14
Summary: Grew crystals and worked on assays.
Semester goal: Find inhibitors that are reproducible.
2 weeks: Purify protein in -80 C and create trays, as well as preparing an assay, plus virtual screening. 2 purification analysis.
Christina 1/22/14
Expression
Final goal: Enzyme assays, finding an inhibitor. Test 5 compounds.
2 week goal: Expression + purification + characterization
Wed - Jan 22nd Progress: Expression completed Goal for Next 2 Weeks: Purification, Characterization, and determine which compounds to order
YopH Project
ToDo List:
see the NotesSBRS_VDSCollaboration
in the VDSStaff GDocs/SBRS-VDScollaboration folder
Researcher: Nicole
Researcher: Anita V Date: 1/22/14 Summary: YopH: Enzyme works and potential inhibitor. Future steps: Test inhibitors to give to other stream, and then test the potential improvements. Ideally crystallize. Work with Jesus. 2 week goal: Purify more YopH, run a confirmation assay. Order: inhibitor.
Researcher: De La O Date: 1/22/14 Summary: L.Mon: Express and test if long protein expression works YopH: Express and purify. Future steps:
2 weeks: Express both L.mon and YopH and test for functional protein.
Wed - Feb 5th Progress: Long protein expression worked on L.mon but was unable to purify it. L.mon was lost in the wash so need to check the pH and to see if it dimerized.YopH was expressed and has gone through nickel column purification. YopH still needs to be fplc due to one contaminating band. Goal for Next 2 Weeks: Start doing Assays on YopH and Retry to express and purify L.mon.
Researcher: Serena Z Date: 1/22/14 Summary: Inhibition assays (4-5), one promising. Substrate is ordered. Future steps: Find a promising inhibitor
2 weeks: Test more inhibitors and order some.
Order: Inhibitors
Researcher: Imran Z Date: 1/22/14 Summary: PP2C of T. gondii. Expression was successful (SDS gel). Future steps: Virtual screening, screen chembridge or maybridge libraries. Screen in-house compounds. Complete inhibition assays.
2 weeks: Reexpress (go big!). Set up virtual screening with in-house or CB306. Utilize pos/neg controls. Researcher: Madeline J/Brendan C Date: 1/22/14 Summary: MDM1 enzyme. Currently virtual screening other libraries. Maybridge library is set up. Complete ICM runs and compare to GOLD. Future steps: Find 5-10 unique scaffolds and complete successful inhibition assays.
2 weeks: Ensure than enzyme is functioning properly (side-by-side comparison). Methacyllin. Researcher: Fin G and Daniel D Date: 1/22/14 Summary: PP2C of L. major. Have already screened CB 306 and InHouse compounds. Tried to express PP2C twice unsuccessfully. Future steps: Virtual screen Chembridge Diversity library. Express and establish enzyme activity on PP2C and identify about 10 compounds for inhibition assay.
2 weeks: Redo expression, purification, and characterization and identify problems if any
Wed - Feb 5th Progress: Completed first round of expression, purification, and characterization of a 1 L culture. Ni-NTA affinity purification was very successful and a distinct band can be seen at 30 kDa. Enzyme assay was performed to test enzyme activity. No enzyme activity was detected for all protein concentrations. Goal for Next 2 Weeks: Re-express a larger 4 L culture. Shorten the transition time between purification and storage. A brief enzyme assay and SDS-PAGE gel will be performed immediately after purification to minimize denaturing effects of short-term storage.
Wed - Feb 5th - April 2nd Progress: Successfully re-transformed LmajSTPP into DH5alpha cells. Grew up small culture, and midi prepped it (90 ng/ul). Successfully transformed into BL21(DE3) cells. Started small culture to express in 4L large culture.
BL21(DE3) - 10 ul
BL21(DE3) - 50 ul
DH5 alpha - 10 ul
DH5alpha - 50 ul
Goal for Next 2 Weeks: Successfully purify protein and conduct more virtual screening
Wed - April 16th Progress: Goal for Next 2 Weeks:
Wed - April 30th Progress: Goal for Next 2 Weeks: Researcher: Grant T Date: 1/22/14 Summary: Successful assay, protein is available. Future steps:Test more inhibitors. Start with testing in-house inhibitors. Complete more virtual screening. 2 weeks: Make more protein Researcher: Oscar Date: 1/22/14 Summary: Positive inhibitor worked Future steps: Crystallize, find a novel inhibitor 2 weeks: Perfect a purification technique and binding assay
Please fill out a progress report in this format every two weeks regarding where you are in your research. In "Summary," briefly explain what you have accomplished in the last two weeks.
We have a UTList Serv that we can communicate when people will be in lab. Anita will add you to the list.
**vds-continuing-researchers**
Bi-Weekly Update Schedule (i.e. every other week, not twice weekly!)
-- copy these lines below to your section
-- update your progress for each 2 week block
-- make a new goal for next 2 weeks
Wed - Jan 22nd
Progress:
Goal for Next 2 Weeks:
Wed - Feb 5th
Progress:
Goal for Next 2 Weeks:
Wed - Feb 19th
Progress:
Goal for Next 2 Weeks:
Wed - Mar 5th
Progress:
Goal for Next 2 Weeks:
Wed - Mar 19th
Progress:
Goal for Next 2 Weeks:
Wed - April 2nd
Progress:
Goal for Next 2 Weeks:
Wed - April 16th
Progress:
Goal for Next 2 Weeks:
Wed - April 30th
Progress:
Goal for Next 2 Weeks:
Joey
1/22/14
Summary: Grew crystals and worked on assays.
Semester goal: Find inhibitors that are reproducible.
2 weeks: Purify protein in -80 C and create trays, as well as preparing an assay, plus virtual screening. 2 purification analysis.
Christina
1/22/14
Expression
Final goal: Enzyme assays, finding an inhibitor. Test 5 compounds.
2 week goal: Expression + purification + characterization
Wed - Jan 22nd
Progress: Expression completed
Goal for Next 2 Weeks: Purification, Characterization, and determine which compounds to order
YopH Project
ToDo List:
see the NotesSBRS_VDSCollaboration
in the VDSStaff GDocs/SBRS-VDScollaboration folder
Researcher: Nicole
Researcher: Anita V
Date: 1/22/14
Summary: YopH: Enzyme works and potential inhibitor.
Future steps: Test inhibitors to give to other stream, and then test the potential improvements. Ideally crystallize. Work with Jesus.
2 week goal: Purify more YopH, run a confirmation assay.
Order: inhibitor.
Researcher: De La O
Date: 1/22/14
Summary: L.Mon: Express and test if long protein expression works YopH: Express and purify.
Future steps:
2 weeks: Express both L.mon and YopH and test for functional protein.
Wed - Feb 5th
Progress: Long protein expression worked on L.mon but was unable to purify it. L.mon was lost in the wash so need to check the pH and to see if it dimerized.YopH was expressed and has gone through nickel column purification. YopH still needs to be fplc due to one contaminating band.
Goal for Next 2 Weeks: Start doing Assays on YopH and Retry to express and purify L.mon.
Researcher: Serena Z
Date: 1/22/14
Summary: Inhibition assays (4-5), one promising. Substrate is ordered.
Future steps: Find a promising inhibitor
2 weeks: Test more inhibitors and order some.
Order: Inhibitors
Researcher: Imran Z
Date: 1/22/14
Summary: PP2C of T. gondii. Expression was successful (SDS gel).
Future steps: Virtual screening, screen chembridge or maybridge libraries. Screen in-house compounds. Complete inhibition assays.
2 weeks: Reexpress (go big!). Set up virtual screening with in-house or CB306. Utilize pos/neg controls.
Researcher: Madeline J/Brendan C
Date: 1/22/14
Summary: MDM1 enzyme. Currently virtual screening other libraries. Maybridge library is set up. Complete ICM runs and compare to GOLD.
Future steps: Find 5-10 unique scaffolds and complete successful inhibition assays.
2 weeks: Ensure than enzyme is functioning properly (side-by-side comparison). Methacyllin.
Researcher: Fin G and Daniel D
Date: 1/22/14
Summary: PP2C of L. major. Have already screened CB 306 and InHouse compounds. Tried to express PP2C twice unsuccessfully.
Future steps: Virtual screen Chembridge Diversity library. Express and establish enzyme activity on PP2C and identify about 10 compounds for inhibition assay.
2 weeks: Redo expression, purification, and characterization and identify problems if any
Wed - Feb 5th
Progress: Completed first round of expression, purification, and characterization of a 1 L culture. Ni-NTA affinity purification was very successful and a distinct band can be seen at 30 kDa. Enzyme assay was performed to test enzyme activity. No enzyme activity was detected for all protein concentrations.
Goal for Next 2 Weeks: Re-express a larger 4 L culture. Shorten the transition time between purification and storage. A brief enzyme assay and SDS-PAGE gel will be performed immediately after purification to minimize denaturing effects of short-term storage.
Wed - Feb 5th - April 2nd
Progress: Successfully re-transformed LmajSTPP into DH5alpha cells. Grew up small culture, and midi prepped it (90 ng/ul). Successfully transformed into BL21(DE3) cells. Started small culture to express in 4L large culture.
Goal for Next 2 Weeks: Successfully purify protein and conduct more virtual screening
Wed - April 16th
Progress:
Goal for Next 2 Weeks:
Wed - April 30th
Progress:
Goal for Next 2 Weeks:
Researcher: Grant T
Date: 1/22/14
Summary: Successful assay, protein is available.
Future steps: Test more inhibitors. Start with testing in-house inhibitors. Complete more virtual screening.
2 weeks: Make more protein
Researcher: Oscar
Date: 1/22/14
Summary: Positive inhibitor worked
Future steps: Crystallize, find a novel inhibitor
2 weeks: Perfect a purification technique and binding assay